Enteroviruses (EVs) (genus Enterovirus, family Picornaviridae) constitute a broad range of pathogens etiologically responsible for a wide range of diseases in both humans and in other animals. Enteroviruses are small RNA viruses that contain positive, single stranded RNA as the genome. Five groups are found within the enteroviruses: coxsackievirus A, coxsackievirus B, echovirus, poliovirus, and the numbered enteroviruses. Most EV infections are asymptomatic or result in only mild symptoms, such as non-specific febrile illness or mild upper respiratory symptoms (for example, the common cold). However, enteroviruses can also cause a wide variety of other clinical illnesses, including acute hemorrhagic conjunctivitis, aseptic meningitis, undifferentiated rash, acute flaccid paralysis, myocarditis, and neonatal sepsis-like disease.
Molecular diagnostic tests to detect EV in clinical specimens usually target highly conserved sites in the 5′ non-translated region (5′-NTR), allowing detection of all members of the genus (Romero, J. R., Arch. Path. & Lab. Med. 123:1161-69, 1999). These tests are genus-specific and provide an EV-positive or EV-negative result but cannot be used to identify the serotype.
Molecular diagnostic tests that target the EV VP1 capsid gene, which correlates with serotype determined by antigenic methods (Oberste et al., J. Virol. 73:1941-48, 1999), can provide both virus detection and identification (Oberste et al., J. Clin. Microbiol. 38:1170-74, 2000 and Oberste et al., J. Clin. Virol. 26:375-77, 2003). However, the identification of serotype, particularly from clinical specimens, is problematic because the virus titer is very low in original specimens. As a result, non-specific amplification can out-compete virus-specific amplification. Additionally, highly degenerate, inosine-containing primers used in diagnostic tests to broaden specificity to include all serotypes (Casas et al., J. Med. Virol. 65:138-48, 2001) often result in non-specific amplification of host cell nucleic acids that obscure the virus-specific product (Rose et al., Nucl. Acids. Res. 26:1628-35, 1998). To overcome these limitations additional molecular diagnostic methods are needed.